重组腺病毒载体AdOX40Ig的构建、表达及相关体外实验

Abstract
Figure/Table
References
Related Citation (2)

Download: PDF (1158 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Objective: To construct and identify recombinant adenovirus vector containing the human OX40-IgG1 fusion gene, then to infect the HL-7702 liver cells to filter out the best infective dose and detect expression of OX40Ig,which lay a foundation for the immune therapy inhibitory after organ transplantation.Methods: Specific OX40 and hIgG1 Fc primers were designed according to GenBank, and subcloned into the pAdTrack-CMV shuttle plasmid after PCR and identification. Retransformed into BJ5183 competent cells with pAdeasy-1 by calcium chloride method. The recombinant plasmid was detected by PCR and DNA sequence analysis and was transfected into 293A cells. The recombinant adenovirus infected HL-7702 liver cells and filtered out the best infective dose. OX40Ig gene expression was detected by indirect immunofluorescence assay. OX40Ig protein expression was detected by ELISA.Results:pTOX40Ig and pAdOX40Ig were constructed successfully according to restriction endonuclease analysis. 293A cells were transfected, and then the purified recombinant adenovirus infected HL-7702 liver cells.10 MOI was the best infective dose.OX40Ig effective expression was detected in HL-7702 liver cells. Conclusion: The recombinant adenovirus vector AdOX40Ig carrying the human OX40-IgG1 fusion gene was successfully constructed, and OX40Ig effective expression was found in HL-7702 liver cells in vitro.

Received: 11 March 2012

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors

Cite this article:

URL:

http://zzs.ujs.edu.cn/xbyxb/EN/ OR http://zzs.ujs.edu.cn/xbyxb/EN/Y2013/V23/I1/12

［1］ Pascual M, Theruvath T，Kawai T, et al. Strategies to improve long-term outcomes after renal transplantation［J］.N Engl J Med,2002,346(8):580-590.

［2］ Bloom RD, Doyle AM. Kidney disease after heart and lung transplantation［J］.Am J Transplant, 2006, 6(4):671-679.

[3] Croft M.Control of immunity by the TNFR-related molecule OX40(CD134)［J］.Annu Rev Immunol, 2010, 28:57-78.

［4］ Cavanagh MM,Hussell T. Is it wise to target the late costimulatory molecule OX40 as a therapeutic target?［J］.Arch Immunol Ther Exp,2008, 56(5):291-297.

［5］ Sugamura K,Ishii N,Weinberg A,et al.Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40［J］.Nat Rev Immunol,2004,4(6):420-431.

［6］ Kinnear G, Wood KJ, Marshall D,et al. Anti-OX40 prevents effector T-cell accumulation and CD8+T-cell mediated skin allograft rejection［J］.Transplantation, 2010,90(12): 1265-1271.

［7］黄一虹,冯洒然,杜冰,等.移植物化学修饰与阻断 OX40-OX40L途径预防异基因骨髓移植小鼠移植物抗宿主病的研究［J］.中华血液学杂志,2009,30(11):735-740.

［8］ Vincenti F. Chronic induction: what^，s new in the pipeline［J］.Contrib Nephrol, 2005 ,146:22-29.

［9］石向华,刘小友,孙煦勇,等.CD40阻断剂对大鼠肾移植急性排斥反应的影响［J］.南方医科大学学报,2011,31(12):2085-2086.

［10］ Lunsford KE, Barbas AS, Brennan TV,et al. Recent advances in immunosuppressive therapy for prevention of renal allograft rejection［J］.Curr Opin Organ Transplant, 2011, 16(4):390-397.

［11］ Sang W,Zhou C,Cheng N,et al. Control of mouse graftversushost disease following allogeneic bone marrow transplantation by blocking the CD28／B7 signaling pathway with lentiviral vector-mediated RNA interference［J］.Immunol Lett, 2011, 136 (2): 194-202.

［12］ Aqarwal A,Newell KA. The role of positive costimulatory molecules in transplantation and tolerance［J］.Curr Opin Organ Transplant,2008,13(4):366-372.

［13］ Sugamura K,Ishii N,Weinberg AD,et al. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40［J］.Nat Rev Immunol,2004,4(6):420-431.

［14］ HonkanenScott M,Johnson J,Hering B,et al. Blockade of OX40 signals enhance survival of xenoislet grafts in spontaneously diabetic NOD mice［J］.Transplant Proc, 2008,40(2):483-485.

［15］ Tavlor L,Schwarz H.Identification of a soluble OX40 isoform:development of a specific and quantitative immunoassay［J］.J Immunol Methods,2001,255(1／2):67-72.